Monoclonal Antibody 3F1H10 Neutralising VHSV (Viral Haemorrhagic Septicaemia Virus)

ABSTRACT

The present invention relates to a non-infectious nucleic acid (RNA and DNA) construct constructed to express a recombinant antibody or antibody fragment in a host cell. The antibody molecule confers protection to the host against a pathogen, allergen or toxin. The host may be any animal including a human.

The present invention relates to a non-infectious nucleic acid (RNA andDNA) construct constructed to express a recombinant antibody or antibodyfragment in a host cell. The antibody molecule confers protection to thehost against a pathogen, allergen or toxin. The host may be any animalincluding a human.

Passive immunization by injection of homologous or heterologousserum-antibodies is routinely used in humans for immunoprophylaxis ofpeople traveling to foreign regions involving risk of exposure to exoticpathogens. In animals a similar strategy may be employed for protectionof valuable specimens, but is generally too expensive for routineveterinary use. Passive immunisation of animals against infectiousdiseases is thus mostly done on an experimental basis with the aim ofstudying the function of structures such as antibodies in vivo andrelating the results to in vitro experiments.

During the recent decade, diverse technologies for the in vitroproduction of antibodies by the use of recombinant DNA technology hasbeen developed. The smallest functional recombinant antibody combiningthe actions of the heavy (H) and light (L) polypeptide chains as in thenative molecule has proved to be the single chain variable-fragmentconstruct (single chain FV). The single chain FV construct is composedof the variable parts of the H and L chains connected by a flexiblespacer region. Such molecules have been used in various studiesincluding virus neutralisation, cancer-immunotherapy and recently alsoin the form of DNA vaccines where plasmids encoding anti-idiotype singlechain FV antibodies have proved able to induce an antigen-specificimmune response. However, direct establishment of protective immunity toinfectious diseases by prophylactic treatment with plasmid DNA carryingsingle chain FV genes encoding protective antibodies has not beendescribed.

An object of the present invention is to provide a non-infectiousnucleic acid construct which can produce an antibody molecule in vivothereby conferring immunity to a disease.

A further object of the present invention is to provide a method ofestablishing immunity against a pathogen.

A yet further object of the present invention is to provide a method oftherapy for animals which have a deficient immune system.

An additional object of the present invention is to provide a method oftherapy for an animal suffering from an allergic reaction or a method ofpreventing an allergic reaction.

For avoidance of doubt it should be noted that the word “animal”includes but is not restricted to mammals including humans.

According to an embodiment of the present invention there is provided anucleic acid construct encoding a recombinant antibody molecule, saidconstruct being adapted for the in vivo establishment of a protectiveimmunity to an infectious disease in an animal.

According to a further embodiment of the present invention there isprovided a nucleic acid construct encoding a recombinant antibodymolecule, said construct is formulated for the in vivo prevention of anallergic reaction to an allergen in an animal.

According to a yet further embodiment of the present invention there isprovided a nucleic acid construct encoding a recombinant antibodymolecule, wherein said construct is formulated for the in vivoprevention of a reaction caused by the presence of a toxic substance inan animal.

The term recombinant antibody molecule encompasses a full size antibody,a single chain variable fragment or any part of an antibody which canrecognise an antigen. In this connection, conveniently the antibodyfragment does not have to be single chain. However, in some embodimentsit is single chain.

It has now been found that the intramuscular injection of a nucleic acidconstruct, in the form of a plasmid, encoding a virus-neutralisingsingle chain antibody fragment can mediate in vivo expression ofantibodies which protect an animal against a possibly lethal exposure toa virus. This has been established in an experimental model whichinvolves a fish rhabdovirus called viral haemorrhagic septicaemia virus(VHSV) in the rainbow trout (Oncorhynchus mykiss) as a host species.

According to a further embodiment of the present invention there isprovided a nucleic acid construct, such as a plasmid, comprising anexpression vector and a gene sequence for heavy and/or light chainvariable domains of an antibody.

Preferably the heavy and light chain variable domains are linked by alinker sequence in order that they form what is known in the art as asingle chain variable-fragment.

It is thought that the antibody fragment as expressed in and secretedfrom a host cell carrying the vector will act with the same specificityas a natural antibody would in the presence of a substance which itrecognises. In this connection, for example, if the heavy and/or lightchain variable domain were derived from a monoclonal antibody raisedagainst dengue virus then if dengue virus infected a host who hadreceived a nucleic construct expressing a single chain variable fragmentproduced from the heavy and light chain of the monoclonal antibody, thefragment would recognise cells infected with the dengue virus or thedengue virus particle itself and bind thereto thereby neutralising orinhibiting the virus and/or giving the host time to mount an immuneresponse against the virus.

In preferred embodiments the expression vector is made for eukaryoticexpression and/or is non infectious. For example, a bacterial plasmid,or a smaller DNA fragment carrying the variable fragment antibody genewithin a eukaryotic expression operon including regulatory elements suchas an enhancer, promoter and polyadenylation signal could be used.Alternatively, stabilised messenger RNA including a positive strandtranscript of the variable-fragment antibody gene with translationsignals may be employed.

The antibody fragment genes can be cloned by any method known to thoseskilled in the art, for example from hybridoma cells or directly fromB-lymphocytes from immunized individuals. Nucleic acid constructsencoding protective antibody fragments can be prepared against anyimportant pathogen/disease causing agent in animals including pathogensagainst which vaccines are not available or have proved insufficient.Furthermore, as a result of veterinary regulations, use of live vaccinesmay not be allowed. In such cases an alternative prophylactic measurewould have to be taken. Such a measure could be the administration ofthe nucleic acid construct of the present invention. A list of possiblepathogens is given below; this list is not intended to be exhaustive.

Viral haemorrhagic septicaemia virus (fish)

Infectious haematopoietic necrosis virus (fish)

Infectious salmon anemia virus (fish)

Infectious pancreatic necrosis virus (fish)

Nodaviruses (fish)

Renibacterium salmoniarum (fish)

Pasteurella (fish)

Ichthyopthtirius mulitifiliis (fish)

NewCastle disease virus (fowl)

Infectious bursal disease virus (fowl)

Bovine respiratory syncytial virus (cattle)

Bovine virus diarrhoea virus (cattle)

Porcine reproductive and respiratory syndrome virus (pigs)

Pseudorabiesvirus (pigs)

Equine herpes virus 1 (horses)

Plasmocytosis virus (mink)

Rabies virus (dogs)

Feline leukemia virus (cats)

Foot and mouth disease (cattle)

Human immune deficiency virus (human)

Hepatitis A virus (human)

Borrelia sp. (human)

Plasmodium sp. (human)

Rabies virus (human)

Epstein-Barr virus (human)

In case of humans with either a congenital or acquired immunodeficiency,vaccines will generally be insufficient. In such cases, administrationof a number of nucleic acid constructs according to the presentinvention encoding antibodies against a broad spectrum of pathogens maybe considered.

For the purpose of prevention of allergic relations induced by IgEresponse, administration of nucleic acid constructs mediating expressionof an allergen-specific recombinant antibody may be used tocompetitively inhibit binding of the allergen to the IgE molecules inthe host. Alternatively gene constructs encoding anti-IgE antibodies maybe used to interfere with the interaction between IgE and mast cells inthe allergic individual.

Administration of antibody gene constructs encoding antibodies to toxinsor venoms can be used for the prophylactic treatment of individualsperiodically being in high risk of exposure to toxic organisms. Thevenoms could, for example, be from snakes or spiders.

Conveniently the construct further comprises a gene encoding a signalsequence for the secretion of the product encoded by the gene sequence.The signal sequence will allow the product of the gene sequence to besecreted from a cell in which the gene has been expressed, into theblood so that the product of the gene sequence can circulate therein.For example, the genes for the signal sequence of either rainbow trouttransforming growth factor beta (TGF-beta), or murine Ig kappa-chain canbe added to the 5′ end of a gene to be administered to the fish. Othersecretion signals, preferably of homologous origin to the host speciesmay be employed. Examples of genes which encode proteins which act assecretion signals include the gene for immunoglobulin heavy and lightchain secretion signals or other glycoprotein secretion signals.Preferably, the secretion signal should include a proteolytic cleavagesite ensuring removal of the signal peptide before secretion of theantibody fragment.

Preferably the construct further comprises a known gene sequence whichencodes a short peptide sequence that can be used to identifytransfected cells. Such a gene sequence can be attached to the 3′ end ofthe gene. Examples of such a sequence include a human kappa light chainconstruct or sequence encoding a six histidine residue. In both cases,an antibody specifically recognising the expressed peptide iscommercially available.

The construct according to the present invention may be delivered by anysuitable method, such as by injection (e.g. intramuscularly), by a sprayon a mucosa surface (e.g. intranasally), by particle bombardment onskin/dermis through use of a gene gun, by electroporation or by uptakeby an animal from an aqueous environment. In this connection, theplasmid may be encased in a liposome for administration to an animal.The construct may be administered to the animal topically, throughinhalation or orally. For oral administration the construct should beprotected from degradation by proper encapsulation.

It is preferred that in a composition or formulation for administrationof the constructs there are present genes encoding the heavy and/orlight chain variable fragments against several different epitopes or anvariable fragment antibody gene expression library against a givenpathogen. In this connection, the various fragments may be provided onone plasmid or they may be provided on several different gene constructswhich are all present in the same formulation or other method ofadministration. In the alternative, each plasmid may have to beadministered separately.

The invention also provides for a method for treating an animal, forexample a mammal or a fish which comprises administering thereto aplasmid or other nucleic acid construct encoding a protective antibodyfragment as previously described.

The invention thus provides for a method of therapy for an animal whichhas a deficient immune system.

The invention also provides for a therapeutic composition comprising theplasmid as previously described and a pharmaceutically acceptablediluent or carrier therefor. The composition may be formulated such thatit is in the form of, for example, a vaccine, dosage form, cream,ointment, liquid or paint.

The invention will now be described by way of illustration only withreference to the following Example and Figures.

FIG. 1 shows a schematic drawing of the pcDNA3 plasmid with a singlechain antibody (ScAb) gene construct inserted downstream of a strongeukaryotic promoter from cytomegalovirus (CMV). PcDNA3 is a commerciallyavailable eukaryotic expression vector (Invitrogen).

FIG. 2 shows a culture of EPC cells (passaged fish cells) transfectedwith a pcDNA3-BU1. BU1 is a ScAb gene construct encoding a recombinantantibody which is able to neutralise the fish pathogenic rhabdovirus,VHSV. BU1 carries a part of the human kappa light chain gene as aresidue or tag. Twelve days after the date of transfection the cellswere fixed and stained immunochemically using horseradishperoxidase-conjugated rabbit antibody to human kappa light chain(HRP-Rabbit anti-kappa) for the detection of cells containing ScAb.These cells give a positive response and are darker than the remainingcells; and

FIG. 3 shows a histological section of muscle tissue sampled from a fishtwelve days after intramuscular injection of pcDNA3-BU1. The section wasstained immunochemically using HRP-rabbit anti-kappa for the detectionof ScAb. Several cells turned out positive (arrow heads) along theregenerating needle track (injection site) arrowed.

Gene Map

The following gene map is the DNA sequence of the construct comprising asingle chain antibody gene (BU1) inserted into E. coli pcDNA3 plasmid(Invitrogen) used in the Example described below. 1 cagtgtgctaacatgagggc agtgtgtttg atgctgactg ccttattgat 51 gctggaatat gtgtgccggagtgaccaggt gcagctgcag gagtcaggac 101 ctggcctcgt gaaaccttct cagtctctgtctctcacctg ctctgtcact 151 ggctactcca tcaccagtgg ttattactgg acctggatccggcagtttcc 201 aggaaataaa ctggaatgga tgggctacat aagctacgac ggtaccaata251 actacaaccc atctctcaca aatcgaatct ccatcactcg tgacacatct 301aagaaccagt ttttcctgaa gttgaaatct gtgactactg aggacacagc 351 tacatattactgtgtaagag ggatctacta tggtaacgac tggtttgctt 401 actggggcca agggaccacggtcaccgtct cctcagaagg caaatcttct 451 ggctctggct ctgaatctaa agtggatgacatcgagctca cccagtctcc 501 tgcctcccag tctgcatctc tgggagaaag tgtcaccatcacatgcctgg 551 caagtcagac cattggtaca tggttagcat ggtatcaaca gaaaccaggg601 aaatctcctc agctcctgat ttatgctgca accagtttgg cagatggggt 651cccatcaagg ttcagtggta gtggatctgg cacaaaattt tctttcaaga 701 tcagcagcctacaggctgaa gattttgtaa gttattactg tcaacaactt 751 tacagtactc cgtacacgttcggagggggg accaagctcg agatcaaacg 801 gactgtggct gcaccatctg tcttcatcttcccgccatct gatgagcagt 851 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataacttctatccc 901 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa951 ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc 1001tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 1051 tacgcctgcgaagtcaccca tcagggcctg agttcgcccg tcacaaagag 1101 cttcaaccgc ggagagtcataagttagata tccat

The BU1 insert (ScAb gene construct) is encoded by nucleotides 10 to1125. The coding region nucleotides are 13 to 1122.

The above identified sequence can be found in the Genebank, theAccession Number is AF302092.

EXAMPLE

Single chain antibody genes were prepared according to the proceduredescribed by McGregor et al; Spontaneous Assembly of Divalent SingleChain Antibody Fragments in E-Coli; Mol. Immunol, February 31(3) pp 219to 226; 1994. In short, the variable domains of the immunoglobulin H andL chain genes were cloned from hybridoma cell lines producing monoclonalantibodies to the fish pathogenic rhabdovirus viral haemorrhagicsepticaemia virus (VHSV). The H and L chain variable domains were linkedby a gene sequence encoding a 14 amino acid linker to generate a singlechain antibody (ScAb) gene. As a tag to allow specific detection, thehuman kappa light chain constant domain gene was included at the 3′ endof the gene. In order to ensure secretion of the ScAb polypeptides ineukaryotic cells, the nucleotide sequence encoding the 20 amino acidsignal peptide of rainbow trout transforming growth factor beta(TGF-beta) was added at the 5′ end of the gene.

The gene construct was inserted by blunt-end ligation into theeukaryotic expression vector pcDNA3 (Invitrogen) in the EcoR I site inthe polylinker downstream of a cytomegalovirus (CMV) promoter (see FIG.1). As a negative control in transfection experiments with cell culturesand immunoprotection trials in fish, the pcDNA3 plasmid without insertwas used. Plasmid DNA was purified from overnight cultures of E. coli byuse of commercial kits for anion-exchange chromatography as recommendedby the supplier (Qiagen).

Other molecular biology procedures used were as followed by Sambrook etal in Molecular Cloning; A Laboratory Manual, Second Addition, ColdSpring Harbor Laboratory, USA, (1989). The variable domain genes from ahybridoma cell line secreting the VHSV-neutralising monoclonal antibody3F1H10 were used. Cloning and sequencing of the variable domain geneshas already been described. In the case of antibody 3F1H10, two aminoacids substitutions were made to the H-chain (Asn35a to Thr and Lys64 toThr). The ScAb carrying the variable domains of antibody 3F1H10 wascalled BU1.

Passaged fish cells designated (EPC) were transfected with an anionictransfection reagent (Superfect, Qiagen). Four to six days aftertransfection cell culture supernatant were harvested and analysed forantibody reactivity to VHSV. After removal of the supernatant, the cellsremaining attached to the bottom of the cell culture wells were fixed in80% cold acetone and stained by immuno-peroxidase using horseradishperoxidase-conjugated rabbit antibody to human kappa light chain(HRP-Rabbit anti-kappa) (DAKO, Denmark) in order to detect cellsexpressing ScAb. The effect of transfection on the susceptibility of thecell cultures to VHSV different doses of live VHSV was examined byadding the different doses to wells with cultures of transfected cellsfour days after transfection and the development of cytopathogeniceffects (CPE) was recorded thereafter.

Injection of Plasmid DNA into Fish

Disease free rainbow trout fingerlings, average weight 4.5 g, wereanaesthetised with 0.001% benzokaine and given two 25 μl injections of20 μg plasmid DNA each, in the epaxial muscles below the dorsal fin. Thefish were afterwards kept in groups of approximately 150 individuals in120-liter tanks supplied with running tap water. The fish were fed adlibitum with commercial fish feed. Mean water temperature was 16° C.Injected plasmid constructs included the pcDNA3 vector without insert,and pcDNA3 carrying the ScAb BU1 gene construct (pcDNA-BU1)respectively.

Immunohistochemical Analysis for Expression of ScAb in Infected Fish

Twelve days after injection of plasmid DNA, 10 fish were sampled foreach plasmid construct. After termination of the fish a section ofmuscle tissue was excised from the site of injection. The tissue wasfixed in 10% phosphate buffered formalin and analysed byimmunohistochemistry. Horseradish peroxidase-conjugated rabbitimmunoglobulin (Ig) to human kappa light chain (HRP-rabbit anti kappa)(Dako, Denmark) was used for detection of expressed ScAb.

Sampling of Plasma from Fish

Blood samples were collected 12 days after injection of plasmid DNA fromfish not exposed to VHSV. Due to the small size of the fish, samplingwas performed with heparin-treated capillary tubes after cutting off theposterior fin of fully anaesthetised fish. The fish were terminatedimmediately afterwards. The blood samples were centrifuged at 5000×g andplasma samples were collected and stored at −80° C. until analysed.

Serological Examination for VHSV-Reactive ScAbs

Supernatant from transfected cell cultures and plasma samples fromDNA-injected fish, were examined for anti-VHSV reactive ScAbs by aplaque-neutralisation (50% PNT) assay and by an enzyme-linkedimmunosorbent assay (ELISA).

The ELISA assay was performed in 96-well microtitre plates coated withpurified VHSV. Bound ScAb's were detected with HRP-Rabbit anti-kappa. Inorder to demonstrate that the virus-neutralising activity detected inthe trout plasma was due to the ScAbs produced by the fish and not bytrout antibodies, two variants of the 50% PNT assay were also applied.One variant included parallel examination of the neutralising activityagainst the virulent VHSV3592B and a neutralisation resistant variant ofVHS 3592B (VHSV DK-3542B) selected by cultivating virus in the presenceof the neutralising Mab 3F1A2 which is highly similar to Mab 3F1H10. Theother variant involved pre-incubation of the trout plasma with rabbitantibodies to human kappa light chain or with rabbit antibodies to troutimmunoglobulin before incubation with virus. The 50% PNT microplateassay was performed as described by Olesen and Jørgensen in Detection ofneutralising antibody to Egtved virus in rainbow trout by plaqueneutralising with complement addition, J. Appl Ichthyol, Volume 2, pages35 to 41.

Immunoprotection Trials in Fish

Eleven days after injection of the plasmid, groups of fish were exposedto (challenged with) the virulent VHSV DK-3592β isolate by immersion inwater containing 100 000 50% tissue-culture infective doses per ml.Challenge was performed in 8-liter aquaria with 25-31 fish in each.Three replicate aquaria was included for each plasmid construct. Deadfish were afterwards daily recorded and collected. Dead fish from alltanks were analysed virologically for the presence of VHSV. Mean watertemperature was 16° C. from the time of injection to immediately beforechallenge. At challenge, the fish were adapted to a water temperature of12° C. and this temperature was kept throughout the 20 day challengeperiod.

Immunochemical Detection of Expressed ScAb in cell Culture and in Fish

It was found that after immuno-peroxidase staining using the HRP-rabbitanti-human kappa, single cells expressing ScAb could be detected in EPCcell cultures transfected with the plasmid construct pcDNA3-BU1 (FIG.2), whereas no positive cells were found in cultures transfected withpcDNA3 without insert. Similarly, expression of ScAb could bedemonstrated in muscle sections from injected fish (FIG. 3). No positivecells were found in fish injected with pcDNA3 without insert.

Interference of ScAbs with Propagation of VHSV in Cell Culture

When monolayers of epithelial cell line of cap cell cultures wereinoculated with VHSV four days after transfection, development ofcytopathogenic effect (CPE) as a result of multiplication of VHSV washighly different in cultures transfected with pcDNA3 compared to cellcultures transfected with pcDNA3-BU1. In the latter case only certainplaques of cells became infected and died and there was no furtherdevelopment of CPE in the 8-day observation period. In contrast, whencultures transfected with pcDNA3 were inoculated, all cells becameinfected and were destroyed within 3-6 days as in a normal propagationof VHSV in EPC cells (Table 1). TABLE 1 Susceptibility of transfectedEPC cell cultures to VHSV Plasmid Construct used for Cytopathogeniceffect upon Transfection inoculation with VHSV* pCDNA3 Completedestruction of cell layer pCDNA3-BU1 Plaques*Concentrations of VHSV: 10²-10³ TCID-50/ml cell culture medium.Detection of ScAbs to VHSV in the Fish

When the plasma from injected fish was analysed by ELISA for ScAbsrecognising VHSV, a strong reaction was found in plasma from fishinjected with pcDNA3-BU1. No reactivity was detected in plasma from fishinjected with pcDNA3 without insert. As indicated in Table 2, thelimited amounts of plasma available made it necessary to perform theanalysis on pools of five individuals. The 50% PNT analysis wasperformed on individual plasma samples. All 10 individuals injected withpcDNA3-BU1 neutralised VHSV, whereas no neutralising activity wasdetected in plasma from fish injected with the pcDNA3 (Table 3). Whenplasma from fish injected with pcDNA3-BU1 was preincubated with Rabbitanti-human kappa before testing in 50% PNT, the neutralising activitywas eliminated, whereas no effect was observed upon pre-incubation withnormal rabbit serum or with rabbit serum to trout Ig (Table 4). Theneutralising activity of a positive trout serum control was unaffectedby pre-incubation with normal rabbit serum and with rabbit anti-humankappa, but was highly reduced upon pre-incubation with rabbit serum totrout Ig (Table 4). As with the parent monoclonal antibody 3F1H10,plasma samples from fish injected with pcDNA3-BU1 could neutralise thevirulent VHSV DK-3592β isolate, but not a neutralisation escape-mutant(not shown). TABLE 2 Antibody reactivity in fish plasma: ELISAReactivity with VHSF Injected (A-496 mm) Fish No. * Plasmid Dilution:1/10 Dilution: 1/80 36529 pCDNA3 0 0 36686 0 0 36844 pCDNA3-BU1 3 116-20 3 1* The plasma samples were analysed in pools of 5 individuals.

TABLE 3 Antibody reactivity in fish plasma: Neutralisation of VHSV FishNo. * Injected Plasmid PNT-titres ** 36534 pCDNA3 <10 36849 pCDNA3-BU1160-640* Plasma samples were analysed individually.** Titres represent the reciprocal value of plasma dilutions reducingthe number of plaques to approximately 50% compared to a control wellwithout antibody/plasma.

TABLE 4 Effect of preincubation of trout plasma with rabbit antibodieson PNT-titres* PNT-titres Rabbit to Injected Normal human chain Rabbitto Fish No. Reagent rabbit kappa trout Ig 21-30 (1 pCDNA3-BU1 640 <40320-640 pool) Positive Killed VHSV >10240 >10240 320 trout serum A7.1*In order to allow detection of neutralising trout antibodies, troutcomplement was included as described above.Infection Trial

When challenged with VHSV DK-3592B 11 days after injection of plasmidDNA, most of the fish injected with pcDNA3-BU1 survived whereas highmortalities were observed among fish injected with pcDNA3 (Table 5).TABLE 5 Protection against VHSV Accumulated mortality 20 days postchallenge (mean of Injected Plasmid triplicate tanks) pCDNAB 81%pCDNA3-BU1  6%

To our knowledge, this is the first report demonstrating establishmentof protective immunity to an infectious pathogen in higher vertebratesby administration of genes encoding pathogen specific single chain FVantibodies. The protective activity of the pcDNA-BU1 construct fullycorrelated with the occurrence of neutralising anti-VHSV ScAbs in theplasma of injected fish, and although involvement of non-specificmechanisms cannot be completely excluded, it appears likely that theproduced BU1 ScAb has been the major cause of protection followinginjection of the pcDNA3-BU1 plasmid DNA. Accordingly, in a laterexperiment including challenge of the fish with a virus isolate notrecognised by the recombinant antibody fragment encoded by pcDNA-BU1, noprotection was obtained.

In contrast to DNA-vaccines, including anti-idiotype vaccines, theadministration of plasmid borne genes in this instance do not involvespecific activation of the immune system in the individual. Theprinciple is simply that single chain FV antibody polypeptides producedby the cells that take up the administered plasmid will be systemicallydistributed by the body fluids and protect the individual if infectionwith the pathogen occurs. This corresponds to the mechanism ofprophylaxis against infectious diseases in humans through administrationof antiserum or immunoglobulin from immunised donors or animals, butwithout side effects such as risk of concomitant transfer of infectiousdiseases or induction of hypersensitivity following repeatedadministrations. In order to avoid the pathogen variability overcomingthe immunity established by the plasmid, practical use may involveadministration of plasmids encoding genes of single chain variablefragments to several different epitopes of the pathogen or single chainFV antibody gene-expression library towards a given pathogen.

The principle of genetic immunoprophylaxis according to the inventioncan be extended to mammals and to humans in particular as it is avaluable tool for transient protection of individuals such as travelersagainst exposure to pathogens or toxins where no efficient vaccines areavailable. Similarly, the invention may be used for induction of thesynthesis of antibodies of a desired specificity for use inimmunodeficient individuals. Also the nucleic acid construct of thepresent invention could be used in individuals that produce an allergicresponse to certain allergens, such as pollen. In this connection,production or induction of antibody fragments to those allergens may beused for prevention of an allergic reaction.

Beside the prophylactic aspects of the invention, plasmid constructscarrying genes encoding pathogen/disease antigen specific single chainFV antibodies are of therapeutic use in certain diseases wherein thehost immune system itself is unable to produce antibodies with thenecessary activity.

1. A pharmaceutical composition for protection of an animal against adisease-causing agent, the composition comprising a non-infectiousnucleic acid construct encoding a recombinant antibody to that agent. 2.A pharmaceutical composition according to claim 1 wherein the animal isselected from a mammal or a fish.
 3. A pharmaceutical compositionaccording to claim 1 wherein the animal has a deficient immune system.4. A pharmaceutical composition according to claim 1 wherein thedisease-causing agent is selected from a pathogen, an allergen or atoxic substance.
 5. A pharmaceutical composition according to claim 1wherein the protection is prophylactic.
 6. A pharmaceutical compositionaccording to claim 1 wherein the encoded recombinant antibody is derivedfrom an antibody raised against the disease-causing agent.
 7. Apharmaceutical composition according to claim 1 wherein the encodedantibody molecule comprises variable domains of immunoglobulin Heavy andLight chain genes linked together by a linker sequence.
 8. Apharmaceutical composition according to claim 1, wherein the nucleicacid construct further comprises a gene sequence encoding a secretionsignal peptide.
 9. A pharmaceutical composition according to claim 1comprising genes encoding antibody molecules to several differentepitopes of the disease-causing agent.
 10. A pharmaceutical compositionaccording to claim 1 comprising a gene-expression library encodingantibodies to the disease-causing agent.
 11. A pharmaceuticalcomposition according to claim 10 wherein the gene expression libraryencodes single-chain antibody molecules to the disease-causing agent.12. A pharmaceutical composition according to claim 1 wherein theencoded recombinant antibody is a virus-neutralising antibody.
 13. Apharmaceutical composition according to claim 12 wherein the encodedvirus-neutralising antibody is single chain molecule.
 14. Apharmaceutical composition according to claim 1 including a nucleic acidconstruct encoding a viral haemorrhagic septicaemia virusVHSV-neutralising monoclonal antibody 3F1H10 with two amino acidssubstituents in the H-chain gene respectively Asn 35a to Thr and Lys 64to Thr and with the secretion signal of rainbow trout transforminggrowth factor (TGF-beta) added to the 5′ end of the gene.
 15. Apharmaceutical composition according to claim 6 wherein when thedisease-causing agent is an allergen the antibody molecule is derivedfrom an antibody raised against IgE molecules.
 16. A pharmaceuticalcomposition according to claim 1 wherein the nucleic acid construct isformed from DNA.
 17. A pharmaceutical composition according to claim 1wherein the composition is in the form of a vaccine, dosage form, cream,ointment, liquid or paint.
 18. A pharmaceutical composition according toclaim 17 wherein the composition is for delivery by injection, spray orgene gun.
 19. A method of conferring protection against a disease causedby a pathogen, an allergen or a toxin on an animal comprisingadministering thereto a pharmaceutical composition of claim
 1. 20.(canceled)